七氟醚后处理通过调节FTO/KCNJ2对缺氧/复氧所致心肌细胞损伤的保护作用

The Protective Effects of Sevoflurane Postconditioning on Hypoxia/Reoxygenation Induced Cardiomyocyte Injury Through Regulating FTO/KCNJ2

目的探讨七氟醚后处理对缺氧/复氧(hypoxia/reoxygenation,H/R)诱导的心肌细胞损伤的影响及机制。方法从基因表达综合数据库(Gene Expression Omnibus,GEO)获取心肌缺血再灌注损伤(myocardial ischemia reperfusion injury,MIRI)相关数据集GSE160516,将差异表达基因KCNJ2纳入本研究。采用qRT-PCR检测KCNJ2在H/R和七氟醚处理的心肌细胞中的表达。RIP实验验证KCNJ2和FTO的结合关系。分别采用MTT法和流式细胞术检测细胞活力和凋亡率,使用乳酸脱氢酶(LDH)检测试剂盒测定细胞的LDH释放量。结果七氟醚后处理可抑制H/R诱导的心肌细胞损伤。相对于Control组,H/R心肌细胞中KNCJ2表达下调,而七氟醚可提高H/R心肌细胞中KCNJ2的表达(均P<0.01)。相对于H/R组,七氟醚组细胞增殖活力提高、LDH释放水平和凋亡率下降,但七氟醚对H/R心肌细胞的保护作用能够被敲减KCNJ2部分逆转(均P<0.05)。FTO能够通过抑制KCNJ2的m6A修饰进而增强KCNJ2的表达;七氟醚可上调FTO的表达进而影响KCNJ2的稳定性和表达水平;敲减FTO能够抑制七氟醚对H/R心肌细胞的保护作用,而该作用可被KCNJ2过表达部分挽救。结论七氟醚后处理通过上调FTO/KCNJ2的表达对H/R诱导的心肌细胞损伤起保护作用。

Objective To investigate the effects of postconditioning with sevoflurane on myocardial cell injury induced by hypoxia/reoxygenation(H/R)and its mechanism.Methods Myocardial ischemia reperfusion injury(MIRI)related dataset GSE160516 was obtained from the Gene Expression Omnibus(GEO),and the dferentially expressed gene KCNJ2 was included in this study.The expression of KCNJ2 in H/R and sevoflurane treated cardiomyocytes was detected by qRT-PCR.RIP was used to verify the binding relationship between KCNJ2 and FTO.The cell viability and apoptosis rate were detected by MTT method and flow cytometry,respectively.Release of LDH in cells was detected by lactate dehydrogenase(LDH)test kit.Results Sevoflurane postconditioning could inhibit H/R-induced cardiomyocyte injury.Compared with control group,the expression of KNCJ2 in H/R cardiomyocytes was downregulated,but sevoflurane could promote the expression of KCNJ2 in H/R cardiomyocyte(both P<0.01).Compared with H/R group,cell proliferation activity was increased,LDH release level and apoptosis rate were decreased in sevoflurane group.The protective effect of sevoflurane on H/R cardiomyocytes could be partially reversed by KCNJ2 knockdown(all P<0.05).FTO could promote KCNJ2 expression by inhibiting m6A modification of KC-NJ2.Sevoflurane could upregulate FTO expression and then affect KCNJ2 stability and expression.K nockdown of FTO inhibited the protective effect of sevoflurane on H/R cardiomyocytes,which could be partially reversed by KCNJ2 overexpression.Conclusion Sevoflurane postconditioning has a protective effect on H/R induced cardiomyocyte injury by upregulating the expression of FTO/KCNJ2.